Saccharomyces cerevisiae NRE1 and IRC24 Encode Paralogous Benzil Oxidoreductases

Irc24p is a benzil oxidoreductase encoded on chromosome IX of Saccharomyces cerevisiae . We identified a putative paralog, Nre1p, encoded 284 bp downstream. Both proteins are small, cytoplasmic, and are 52% identical (70% similar). PANTHER and PFAM analysis of the amino acid sequences and rigid pairwise structure alignment predicted a conserved active site and Rossmann folds in both, implicating NADH or NADPH as likely cofactors. We purified hexahistidine-tagged Irc24p and Nre1p. Both proteins catalyze the reduction of the diketone benzil with similar kinetics and a preference for NADPH. This is the first demonstration of in vitro function for Nre1p.


Description
Nre1p is a 254 amino acid protein encoded by the YIR035C open reading frame (ORF) on chromosome IX of Saccharomyces cerevisiae (Fig. 1A). The Saccharomyces Genome Database (SGD) presently characterizes Nre1p as a so-called "orphan" protein, whose function is unconfirmed (Chervitz, 1999). High-throughput protein expression data confirm that Nre1p is cytoplasmic (Kumar, 2002;Huh, 2003).
BLASTP analysis of Nre1p indicated that a 263-amino acid protein encoded 284 bp upstream, Irc24p (Fig. 1A), shares significant identity (52%; Fig. 1B). Irc24p was characterized as a stereoselective benzil reductase some 20 years ago (Maruyama, 2002). Nre1p and Irc24p share cofactor and substrate binding residues (Fig. 1B, yellow) and similarity throughout. We analyzed the sequences of Nre1p and Irc24p using bioinformatic databases including PANTHER, PFAM, and the National Library of Medicine's Conserved Domain Database (CDD), which placed both proteins in the short-chain dehydrogenases/reductases family. The CDD predicted Rossmann folds in both proteins (Fig. 1B, dark purple; Lu, 2020). Nre1p shares 46% amino acid identity with characterized oxidoreductase KRED1-Pglu from the non-conventional budding yeast Ogataea glucozyma and a 28% identity with SDR family ketoreductase from the Gram-negative bacterium Serratia marcescens.
The Protein Data Bank (PDB; https://www.rcsb.org/) currently reports two crystal structures (PDBID: 3KZV and 6UHX) and one computational structure (AlphaFold: AF_AFP40579F1) for Nre1p, though none of these structures is published elsewhere. Rigid pairwise structural alignment of the 100% sequence-identical Nre1p structures 3KZV and 6UHX showed a template modeling score (TM-score) of 0.84 and a root mean square deviation (RMSD) of 2.81 due to divergences limited to unstructured regions. The 6UHX structure has 253/254 modeled residues, compared to 249/254 for 3KZV, though the latter reports a better resolution (2.00 vs. 2.75 Angstroms). We selected 6UHX (Fig. 1C) for additional pairwise comparisons, as it aligned more closely to homologous proteins.
We used AlphaFold to predict a very high-confidence structure for Irc24p, as there is no experimentally determined structure available (Fig. 1D). Only five C-terminal residues (259-263) had confidence scores less than 90/100. We then conducted rigid pairwise structural alignments of Irc24p and Nre1p to determine the extent to which these proteins bear similar threedimensional structures (Fig. 1E). Both the RMSD and TM-Score were close to 1, indicating a near-perfect alignment between the two structures. Additionally, we aligned Nre1p with potential homologues identified in the bioinformatic searches: KRED1-Pglu from Pichia glucozyma and SDR ketoreductase from Serratia marcescens. All structures are consistent with the short-chain dehydrogenase family, benzil reductases, and sepiapterin reductases (Mistry, 2020;Mi, 2020). Although KRED1-Pglu and SDR-KR were each co-crystallized with NADP, structural alignments confirmed Nre1p as a structural homolog (Fig.  1E).
Pairwise structural alignments of the crystal structure of Nre1p and predicted structure of Irc24p also confirmed the signature Rossmann fold (Fig. 1B and 1C, dark purple), which consists of six-stranded, extended β-sheets between a set of α-helices (Rabuffetti, 2020). This structure is well conserved in short-chain dehydrogenase and sepiapterin reductase proteins to utilize FAD, NAD+, or NADP as a coenzyme for redox reactions with diketone compounds (Wu, 2020;Persson, 2002;Bae, 2021). We wished to determine the preferred coenzyme and kinetics of Irc24p and Nre1p with a benzil diketone substrate. We cloned, expressed, and purified hexahisidine-tagged, full-length Nre1p and Irc24p, confirming purity using SDS-PAGE (Fig. 1F, 1G). We tested in vitro enzyme activity with benzil and either NADH or NADPH as a coenzyme in the predicted reduction reaction (Fig. 1H-I). Both enzymes were active with both coenzymes but showed a two-fold preference for NADPH (Fig. 1I). Kinetic analysis showed similar apparent Michaelis constant (Km) values for Nre1p and Irc24p and an apparent specificity constant about half of that of KRED-Pglu (Contente, 2016). Thus, Nre1p is a benzil reductase in vitro and likely paralog of Irc24p.

Bioinformatic Analysis
BLASTP was used to identify paralogous/homologous proteins within and outside S. cerevisiae. Amino acid sequences were aligned and depicted using CLUSTAL Omega. Irc24p and Nre1p were subjected to genome-scale phylogenetics using PANTHER , PFAM (Mistry, 2021), and Conserved Domain Database (CDD; Lu, 2020) to predict protein domains and membership in protein families.

Protein Expression, Purification, and Analysis
Freshly transformed BL21(DE3)PlysS E. coli cells (Novagen) were grown to saturation at 37˚C with shaking (250 RPM) in LB broth with 50 µg/ml kanamycin and 34 µg/ml chloramphenicol. Cultures were diluted 1:50 in LB broth with 50 µg/ml kanamycin and induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) after reaching an OD ~0.6 (mid-log phase). Small analytical samples were collected at 2, 4-, 6-, 8-, and 24-hours post-induction for expression optimization. Samples were prepared with Laemmli buffer and beta-mercaptoethanol plus brief sonication and separated using a Mini-PROTEAN TGX 4-20% gradient SDS-PAGE gel (BioRad) stained in InstantBlue Commassie Protein Stain (Abcam). Cells from 100 ml preparative cultures collected were collected by centrifugation at 4˚C (3,000 x g for 20 min) and frozen at -20˚C until purification. Cell pellets were resuspended with 500 uL of Bacterial Protein Expression Reagent (B-PER; Thermo Scientific) with Roche EDTA-free protease inhibitors (Roche Diagnostics), lysozyme (0.2 mg/mL), and DNAaseI (1 U/uL). Lysate was incubated for 15 minutes at room temperature while mixing. The lysate was sonicated on ice three times for five seconds (setting 3) using a 550 Sonic Dismembrator (Thermo). The lysate was cleared by centrifugation at 15,000 x g for five minutes at 4 o C. The cleared supernatant was added to prepared Ni-NTA agarose beads and incubated for 45 minutes at room temperature according to the manufacturer's instructions (Invitrogen; R90110). The beads were washed three times in wash buffer (10 mM Tris-HCl [pH 8.0], 100 mM KCl, 0.1 mM EDTA, 20 mM imidazole) and eluted twice in elution buffer (10 mM Tris-HCl [pH 8.0], 100 mM KCl, 0.1 mM EDTA, 200 mM imidazole). The eluted sample was dialyzed into 50 mM Tris-HCl, [pH 8.0] using 16 mm dry SnakeSkin 3.5K MWCO (Thermo). Sterile 100% glycerol was added 1:1 and proteins were stored briefly at -20 o C. Bradford assays quantified final yields of 9.46 uM and 9.13 uM, IRC24 and NRE1, respectively.

Oxidoreductase Assays
The enzyme activity assay contained purified IRC24 or NRE1 (1.2 µM), benzil (0.5 mM, 2.5 mM, 5 mM) in ddH 2 O, 0.075 mM NAD(P)H and 10 mM Tris-HCl [pH 8.0]. The mixture was equilibrated to 30 o C for five minutes before adding substrate. Like the previously described method (Maruyama et al., 2002), the reductase activities of IRC24 and NRE1 were spectrophotometrically assayed at 30 o C, and a decrease in absorbance was followed at 340 nm. Enzyme kinetic studies were repeated with these conditions. The enzyme kinetic constants were determined from quadruplicate samples by assuming apparent Michaelis-Menten kinetics using Lineweaver-Burk analysis.